First, canines with Staphylococcus aureus (S. aureus) pneumonia were randomized in a blinded fashion for exchange-transfusion with either 7- or 42-day-old canine commercially available universal donor blood (80 mL/kg in 4 divided doses). These experiments were designed to test the extremes of storage age and transfusion volume. Older blood transfusion increased mortality, the arterial-alveolar oxygen gradient and histological lung damage. Older blood resulted in increased in vivo hemolysis, releasing free iron (in the form of non-transferrin bound iron) and CFH and decreasing haptoglobin plasma levels. Consistent with the vasoconstrictive effect of CFH, older blood increased both systemic and pulmonary pressures. This was the first randomized blinded animal trial showing blood transfused at end of storage period can increase mortality during infection Next, we investigated the effect of increasing bacterial doses and severity of infection on the risks associated with age of blood transfused in this canine model. Without bacterial challenge, levels of CFH and NTBI were significantly higher with older versus fresher blood transfusion but there were no significant differences in measurable injury. With higher-dose bacterial challenge, the elevated NTBI levels declined more rapidly and to a greater extent after transfusion with older versus fresher blood, and older blood was associated with significantly worse shock, lung injury, and mortality. These data suggest that transfused older RBCs increases the risks specifically from infection in septic subjects and define an infection dose-response We wondered whether transfusion of older RBCs would cause similar adverse effects during shock and inflammatory injury without infection. Therefore, animals were transfused similar quantities of either older (42-day) or fresher (7-day) stored universal donor canine RBCs 2.5 hours after undergoing controlled hemorrhage producing shock. With older transfused RBCs, CFH and NTBI levels increased, but lung injury declined and there was a trend toward lower mortality (18% vs. 50%). Interestingly, the increased levels of CFH with older blood transfusion were associated with an improved hemodynamic response to hemorrhage-reperfusion, with lowered exogenous norepinephrine requirements and cardiac outputs. This hemodynamic effect is consistent with the ability of CFH to scavenge NO causing vasoconstriction. These preclinical data suggest that, whereas iron derived from older RBCs promotes bacterial growth worsening septic shock mortality during infection, release of CFH and NTBI during hemorrhagic shock is not necessarily harmful We next conducted a blinded randomized controlled study of RBC washing in this canine model of transfusion injury. Washing older units of blood improved survival rates, shock score, lung injury, cardiac performance and liver function, and reduced levels of NTBI, possibly by lysing and washing away older cells and supernatant. In contrast, washing fresh blood worsened all these same clinical parameters and increased CFH levels. Our data suggest that fresh blood should not be washed routinely because washing induces sub-lethal membrane damage to the RBC and, in a setting of established infection, washed RBCs are prone to lyse, release CFH, and result in worsened clinical outcomes. However, if older blood must be used during established infection, washing prevents elevations in plasma circulating iron and improves survival and lessens multiple organ injury We next examined the results of altering volume, washing, and age of RBCs. Animals were transfused with increasing volumes of old or fresh blood either unwashed or washed RBCs with increasing storage age. Our preclinical data suggest that any volume of older blood potentially increases risks during established infection. In contrast, even massive volumes of fresh blood resulted in minimal CFH and NTBI levels and risks. In contrast to the extremes of storage, washing blood stored for intermediate ages does not alter risks of transfusion or NTBI and CFH clearance No studies have been performed comparing intravenous (IV) iron with transfused red blood cells (RBCs) for treating anemia during infection. During canine experimental bacterial pneumonia, treatment of mild anemia with IV iron significantly increased free iron levels, shock, lung injury, and mortality compared to transfusion of fresh RBCs. This was true for iron preparations that do or do not blunt circulating free iron level elevations. These findings suggest that treatment of anemia with IV iron during infection should be undertaken with caution Transfusion and bacterial sepsis are both independent potential sources of elevated plasma CFH. Increased CFH levels after transfusion and during sepsis have both been associated with worse outcomes in animal models and humans. Haptoglobin binds CFH and promotes its clearance. In a series of studies using a commercially fractionated plasma-derived haptoglobin concentrate, we found that excess haptoglobin improves outcomes in both transfused, and, unexpectedly, in non-transfused septic animals. These findings suggest that administration of haptoglobin may be an effective clinical therapy for managing sepsis and septic shock. Storage temperature is a critical factor for maintaining red-blood cell (RBC) viability, especially during prolonged cold storage. The target range of 1-6C was established decades ago and may no longer be optimal for current blood-banking practices. We completed a study investigating the role of storage temperature and showed the lower bounds of the regulated storage temperature range resulted in greater recovery of transfused RBCs and less hemolysis with release of potentially harmful byproducts such as cell free hemoglobin and iron. Storage at refrigeration temperatures closer to 2C may result in a better product for transfusion and more accurate for chromium RBC viability testing. This study shows that current blood storage practice standards may not adequately account for an important interaction between storage time and temperatures that may confound chromium RBC viability testing and prove clinically relevant In both human septic shock and our canine Staphylococcus aureus pneumonia model, cell-free hemoglobin (CFH) levels are elevated. Presumably, virulent bacteria produce factors which lyse red blood cells (RBCs), liberating CFH. Elevated CFH could scavenge nitric oxide (NO) resulting in endothelial damage and vasoconstriction. The iron in CFH possibly serves as an essential nutrient promoting bacterial growth or contribute to oxidative tissue injury or augments inflammation. Indirect evidence suggests both CFH-NO scavenging and Iron- related mechanisms could cause injury during infection. In our canine pneumonia model, transfusion of RBCs results in elevated CFH and iron levels and increased lethality of S. aureus. This is clinically important since elevated CFH levels in human septic shock are associated with a worse outcome and RBC transfusion is common in patients with sepsis. Human haptoglobin concentrate (Hp) infusion in our canine pneumonia model resulted in the clearance of both iron and CFH, as Hp complexes with CFH to produce a large molecule which remains intravascular until it is cleared by the liver. Independent of receiving RBC transfusion, Hp infusion in septic animals resulted in improved shock, lesser lung injury, and lower mortality. This study is intended to identify the specific mechanism(s) by which hemolysis increases injury in septic shock and how Hp is beneficial.